Understanding the kinetic differences between the Kidney-Type Glutaminase and Glutaminase C isoforms

• Grant number: 11/15107-0
• Support type: Scholarships in Brazil - Master
• Effective date (Start): March 01, 2012
• Effective date (End): December 31, 2013
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: Amanda Petrina Scotá Ferreira
• Home Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia, Inovações e Comunicações (Brasil). Campinas , SP, Brazil

Abstract

The versatile use of the amino acid glutamine by cells, especially for the purposes of biosynthetic activities, makes it the one of the most consumed extracellular nutrients by tumors. In normal differentiated cells, the first step in its metabolism, i.e., conversion of glutamine to glutamate plus an ammonium ion, is performed mainly by three mitochondrial glutaminase isozymes: the Kidney-type glutaminase (KGA) and its splicing variant Glutaminase C (GAC), dependent on inorganic phosphate for its activation and the Liver-type Glutaminase (LGA). Ongoing structural and biochemical studies in our laboratory have provided clues towards the structural determinants for the phosphate-dependent activation mechanism of mammalian GAC, based on the tetramerization-induced lifting of a "gating loop", which controls substrate accessibility to the active site. We showed that phosphate binds inside the catalytic pocket, resulting in allosteric stabilization of tetramers and at the same time facilitating substrate entry by outcompeting with the catalysis product, glutamate, thus guaranteeing enzyme cycling. Even though GAC and KGA have identical glutaminase domains we also documented GAC as the most efficient of the mammalian glutaminases in hydrolyzing glutamine in the presence of inorganic phosphate, possibly due to its facilitated tendency to oligomerize. In this regard, we will now seek a better understanding of the oligomerization profile of the two isoforms, by determining the dissociation constants of its tetramers by fluorescence anysotropy, as well as explore the importance of the C-terminus region of GAC for its activity and oligomerization profile. Additionally, we will try to provide a better description of the movements in the gating loop under different ligand-bound and oligomeric states (dimers and tetramers) and how it affects catalysis by site-directed mutagenesis, x-ray crystallography and electron spin resonance.